Project Abstract/Summary Our underlying hypothesis is that, unlike human classical and other non-classical MHC class I proteins, HLA- F ligand recognition is focused on glycans, either as components of oligosaccharides, proteo/peptidoglycans, or glycoproteins/glycopeptides ? representing a paradigm shift in current understanding. This hypothesis is based on a rigorous reevaluation of recent, otherwise seminal crystallographic and mass-spec results (1). Echoing FOA PA-19-066, our long-term overall goal is ?to characterize antigen processing and presentation [?] of novel peptidic and non-peptidic ligands presented by [HLA-F], and to determine the contribution of these unique antigenic ligands to: protective immune responses to infectious pathogens and/or vaccines; pathogen- associated immune pathogenesis; and/or in the induction/progression or prevention of immune-mediated dis- eases.? Our first steps towards accomplishing this goal in this R21 will be to: (a) identify physiologically- relevant ligand/s for HLA-F using rigorous biochemical approaches, focusing on glycopeptides and glycoconju- gates; and (b) determine conditions for growing diffraction-quality co-crystals to support future crystallographic structure determinations. These results will comprise the necessary preliminary results to support follow-on grant applications to structurally, biologically, and functionally validate candidate HLA-F glycoligands. We will achieve these goals, and rigorously test our hypothesis, through the following Specific Aim: we will use our novel ARTEMIS mass-spec peptide discovery platform, glycan arrays, and fragment screening ap- proaches, to fully parse HLA-F ligand specificity for glycosylated ligands. Candidate ligands will be used to screen crystallization conditions to support follow-on crystallographic studies to eventually structurally characterize recognition mechanisms to fully parse specificity. Rigor will ultimately be achieved through careful biochemical and structural validation, and the likelihood of success will be maximized by incorporating multiple, parallel ap- proaches and experimental methods into the proposed scope to overcome potential confounders and pitfalls. Significance (from PA-19-066): ?The classic understanding of antigen processing and presentation begins with a protein fragment (peptide) associating with MHC molecules. [?] These long-accepted paradigms regard- ing antigen processing and presentation and T cell recognition are incomplete. Approximately 10% of antigenic peptides can be derived from unconventional sources [?]. Additionally, the antigen components that trigger non- classical MHC I restricted CD8 T cells and unconventional or innate T cells include lipids and small-molecule metabolites but are not well characterized. [?] The objective of this research program is to promote the discovery of unique antigenic ligands (peptidic and non-peptidic) and understand the immune responses to these ligands: peptidic and non-peptidic ligands presented by non-classical MHC molecules: [HLA-F].?